The severity of sickle cell disease and beta-thalassemia is ameliorated under conditions where individuals produce high levels of fetal hemoglobin. It follows that a rational therapy for these diseases would be to increase fetal globin levels in patients where this does not otherwise occur. To this end, the immediate goal of the research is a better understanding of the mechanisms regulating fetal and adult globin levels in adults. At least three regions of DNA sequences outside of the globin gene transcription unit have been identified which have important roles in regulating fetal gamma- and adult beta-globin gene expression. These are locus activation region (LAR) sequences flanking the beta-globin gene cluster (which contains the two gamma-globin genes), gamma- and beta-globin gene promoter elements, and enhancer elements 3' to the gamma- and beta-globin genes. We plan to study the effects of these sequences on the expression of transferred gamma- and beta-globin genes. In particular, we will determine whether individual gamma- and beta-globin genes can compete for activation by LARs and enhancers thereby influencing their relative expression. An integral set of experiments will be to determine causality of naturally-occurring mutations linked to elevated fetal globin production in otherwise normal adults (the nondeletion types of hereditary persistence of fetal hemoglobin). We will accomplish these aims by retroviral transfer of globin regulatory sequences and globin genes into early erythroid progenitors (burst forming units-erythroid or BFU-E) isolated from human peripheral blood. BFU-Es carrying the transferred gene will be cultured under serum-free conditions to produce mature erythroblasts expressing physiological levels of gamma-globin. Expression of the transferred globin genes will be measured in RNA isolated from the erythroblasts and compared to endogenous globin gene levels. Retroviral transfer of globin genes into human erythoblasts will provide information on human globin gene regulation unobtainable in other systems and will also directly help develop gene therapy.